The background of the invention provides the information on various antibody constructs containing the VHH domain.
PCT/EP2008/066368 publication describes antibodies that comprise separate variable domains linked with Fc-fragment. Nano-bodies can be used as variable domains with Fc obtained from IgE type antibodies. Said domain and Fc fragments can be connected through a linker located in the hinge domain.
Patent application US 2009/0202979 disclosed antibodies comprising complete VHH antibodies or parts thereof directly connected to the constant regions of human antibodies.
In addition, amino acid substitutions are known that affect the physical-chemical and biological properties of antibodies.
For example, application US 20110028348 describes the heavy chain variable domains wherein amino acid substitutions were introduced in positions 35, 45, 47, 93-100 and 100a to improve the hydrophilic properties of the antibody obtained.
Now, the methods have been developed to optimize the structure of isolated VHH and VH mono-domains in order to reduce the immunogenicity and improve the aggregation stability thereof.
Thus, Vincke at al. [22] have found that Glu-49→Gly and Arg-50→Leu substitutions in characteristic amino acids result in the obtainment of a new domain that is more stable yet less soluble. Other substitutions in the framework region FR-2 Phe-42→Val and Gly/Ala-52→Trp are crucial for antibody affinity to the antigen due to re-orientation of H3-loop, so that the dissociation constant increases 6-10-fold (6.85·10−3 l/sec). Phe-42→Val substitution caused the reduced stability of antibodies obtained. The substitution of Gly-49 and Leu-50 in VH-sequence resulted in the lower stability of the domain, while Glu-49 and Arg-50 humanization in VHH allows obtaining the stable variable domains.
It is known from the literature, that in the presence of short HCDR3 regions neutralizing the shading effect of the conformation of classical VHH, and upon introduction of VH-characteristic Trp-47→Gly-47 substitution as well as Tyr-37→Val-37, Glu-4→Gly-44 and Arg-45→Leu-45, the isolated VHH domains can regenerate the ability to bind with VJ domain [24].
The relationship between the increased aggregation stability of therapeutic antibodies of classical IgG structure and the reduced immunogenicity thereof was demonstrated in multiple studies and summarized in the review by Hermeling et al., 2004 [25]. Yet there were no antibodies revealed that comprise VHH domains but were linked to the variable domains of the light chains within the full-size human IgG.
Therefore, there is a need for development of a new format of antibodies that would have improved stability and affinity, good expression and low immunogenicity
Besides, no approaches were earlier described with regard to the development of such molecules that would be easy to obtain, have improved aggregation stability, increased affinity and high expression level in the mammal cell culture.
According to previously mentioned, this invention is the first to describe antibodies comprising VHH-derivatives that are able to bind to variable domains of the light chains of the full-size human IgG, which results in the formation of a construct that is similar to the native one (and, hence, having low immunogenicity) but has improved aggregation stability, increased affinity, and a structure of a therapeutic monoclonal antibody.